Network Pharmacology and Molecular Modeling to Elucidate the Potential Mechanism of Neem Oil against Acne vulgaris

Acne vulgaris is a common skin disorder with a complicated etiology. Papules, lesions, comedones, blackheads, and other skin lesions are common physical manifestations of Acne vulgaris, but the individual who has it also regularly has psychological repercussions. Natural oils are being utilized more and more to treat skin conditions since they have fewer negative effects and are expected to provide benefits. Using network pharmacology, this study aims to ascertain if neem oil has any anti-acne benefits and, if so, to speculate on probable mechanisms of action for such effects. The neem leaves (Azadirachta indica) were collected, verified, authenticated, and assigned a voucher number. After steam distillation was used to extract the neem oil, the phytochemical components of the oil were examined using gas chromatography–mass spectrometry (GC-MS). The components of the oil were computationally examined for drug-likeness using Lipinski’s criteria. The Pharm Mapper service was used to anticipate the targets. Prior to pathway and protein–protein interaction investigations, molecular docking was performed to predict binding affinity. Neem oil was discovered to be a potential target for STAT1, CSK, CRABP2, and SYK genes in the treatment of Acne vulgaris. In conclusion, it was discovered that the neem oil components with PubChem IDs: ID_610088 (2-(1-adamantyl)-N-methylacetamide), ID_600826 (N-benzyl-2-(2-methyl-5-phenyl-3H-1,3,4-thiadiazol-2-yl)acetamide), and ID_16451547 (N-(3-methoxyphenyl)-2-(1-phenyltetrazol-5-yl)sulfanylpropanamide) have strong affinities for these drug targets and may thus be used as therapeutic agents in the treatment of acne.


Introduction
Acne vulgaris, one of the most prevalent skin conditions, is a chronic, self-limiting inflammatory disorder of the pilosebaceous unit. There are several contributing elements to the complex pathophysiology of acne. The Gram-positive bacterium Cutibacterium acnes, formerly known as Propionibacterium acnes, is a significant contributing element. Cutibacterium acnes causes Acne vulgaris to appear throughout adolescence when dehydroepiandrosterone (DHEA) is naturally circulating in the blood. It is a fairly common skin condition that typically affects the face but can also affect the upper arms, torso, and back [1,2]. It can appear with inflammatory and non-inflammatory lesions, papules, comedones, blackheads, and other skin growths.
Males are more likely than females to develop acne [3]. Populations in urban areas are more affected than those in rural areas. Approximately 20% of those who are impacted experience severe acne that leaves scars [3]. It seems that some races are more impacted than others. Severe acne is more common in Asians and Africans, whereas mild acne is more prevalent in white people. People with a darker complexion in general also tend to acquire hyperpigmentation. Neonates can also have acne, but it typically goes away on its own [4].
For mild to moderate acne, topical therapy is the usual course of action. Nowadays, several medicines with complementary actions are routinely used to boost the success of therapy [5] because of the complex pathophysiology of acne. Local skin irritation was linked to the majority of side effects based on this topical approach, which were typically mild to moderate in intensity, intermittent, and rarely resulted in therapy discontinuation [6]. Natural oils are now being tested more and more in the treatment of acne and other skin conditions. Some of these oils are selected for acne treatment based on their antibacterial and/or anti-inflammatory properties.
The neem plant, Azadirachta indica, a member of the Meliaceae family, produces an oil that is recognized to have antibacterial and anti-inflammatory characteristics as well as the capacity to strengthen the immune system [7,8]. This offers a helpful justification for the potential topical management of acne and helps to stop lesions from returning, leaving the skin healthy [9]. While some oils are only non-comedogenic and will not clog pores (which can result in acne), others actually have natural therapeutic capabilities that can lessen the signs of acne and possibly prevent future breakouts. Neem oil is one of these oils, and it has been used for many years to treat skin issues including acne [10]. The breadth of the scarce research that has been completed on neem oil and the treatment of Acne vulgaris is inadequate. Therefore, a novel approach or idea may be required to enable us to fully examine the mechanism behind neem oil's anti-Acne vulgaris effects.
The purpose of this study is to use network pharmacology to screen and predict potential processes through which neem oil may function to treat acne. The probable molecular mechanism of neem in treating Acne vulgaris will be investigated by building the "drug-component target-pathway-disease" interactive network, using GO and KEGG pathway enrichment analysis, and molecular docking methodology. Our findings might serve as a guide for further fundamental experimental study.

GC-MS Analysis
As shown in Figure S1 and Table S1 (Supplementary Materials), the results show that a total of 39 substances were discovered in the Azadirachta indica oil. Cyclohexane was a significant phytoconstituent, with a peak area of (108.73%).

Screened Neem Oil Compounds Targets and Acne vulgaris Disease Targets
A list of the 39 phytocompounds discovered in neem oil was compiled using the PubChem database (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 4 January 2023). Ten compounds were discovered that met the drug-likeness screening requirements based on Lipinski's rule of five (Lipinski's rule of five), molecular weight < 500 KD, hydrogen bond donor < 5, hydrogen bond acceptor < 10, TPSA < 140 Å, and clogP < 5 ( Figure 1).
Target prediction was performed using PharmMapper, and the findings were chosen for further study if the norm fit was greater than 0.6. Of the ten (10) compounds, 309 targets were found. Additionally, the GeneCards database revealed 570 targets of Acne vulgaris. Venn diagrams were created using the targets of neem oil and Acne vulgaris, and the 17 genes that overlapped served as possible targets for neem oil's anti-Acne vulgaris effects ( Figure 2). Target prediction was performed using PharmMapper, and the findings were chosen for further study if the norm fit was greater than 0.6. Of the ten (10) compounds, 309 targets were found. Additionally, the GeneCards database revealed 570 targets of Acne vulgaris. Venn diagrams were created using the targets of neem oil and Acne vulgaris, and the 17 genes that overlapped served as possible targets for neem oil's anti-Acne vulgaris effects ( Figure 2).

GO and Pathway Analyses of the DEGs Associated with Acne vulgaris
In total, 1665 DEGs were found in 6 Acne vulgaris patients and 12 normal samples, including 944 upregulated and 721 downregulated genes ( Figure 3). An additional GO analysis was used to identify potential molecular processes for the DEGs. These genes

GO and Pathway Analyses of the DEGs Associated with Acne vulgaris
In total, 1665 DEGs were found in 6 Acne vulgaris patients and 12 normal samples, including 944 upregulated and 721 downregulated genes ( Figure 3). An additional GO analysis was used to identify potential molecular processes for the DEGs. These genes were highly abundant in "enzyme binding", "muscle cell proliferation", "reaction to oxygencontaining molecule", "response to wounding", "response to organic substance", and "regulation of cytokine production" under the heading biological process (BP) ( Table 1). These genes were significantly involved in "non-membrane spanning protein tyrosine kinase activity", "protein tyrosine kinase activity", "protein serine/threonine/tyrosine kinase activity", "signaling receptor binding", and "cytokine receptor binding" according to the molecular function (MF) category (Table 1). Acute megakaryocytic leukemias, adult classical Hodgkin, and lymphoma were identified in the KEGG pathway study with the enriched genes (Figures 4 and 5). The most important route among these was "Adult Classical Hodgkin" which comprised seven genes ( Figure 4). The DEGs' PPI networks showed that some genes, including MMP13, TGFB2, IL2, STAT1, JAK2, SRC, F2, F3, and CSK, were very strongly connected ( Figure 6). A total of 4 of the 1665 DEGs perfectly overlapped the 17 probable targets for neem oil's anti-Acne vulgaris effects ( Figure 7). Since these four (4) genes were overexpressed in Acne vulgaris (Table 2), inhibitors will be needed as a treatment.

GO and Pathway Analyses of the DEGs Associated with Acne vulgaris
In total, 1665 DEGs were found in 6 Acne vulgaris patients and 12 normal samples, including 944 upregulated and 721 downregulated genes ( Figure 3). An additional GO analysis was used to identify potential molecular processes for the DEGs. These genes were highly abundant in "enzyme binding", "muscle cell proliferation", "reaction to oxygen-containing molecule", "response to wounding", "response to organic substance", and "regulation of cytokine production" under the heading biological process (BP) ( Table 1). These genes were significantly involved in "non-membrane spanning protein tyrosine kinase activity", "protein tyrosine kinase activity", "protein serine/threonine/tyrosine kinase activity", "signaling receptor binding", and "cytokine receptor binding" according to the molecular function (MF) category (Table 1). Acute megakaryocytic leukemias, adult classical Hodgkin, and lymphoma were identified in the KEGG pathway study with the enriched genes (Figures 4 and 5). The most important route among these was "Adult Classical Hodgkin" which comprised seven genes ( Figure 4). The DEGs' PPI networks showed that some genes, including MMP13, TGFB2, IL2, STAT1, JAK2, SRC, F2, F3, and CSK, were very strongly connected ( Figure 6). A total of 4 of the 1665 DEGs perfectly overlapped the 17 probable targets for neem oil's anti-Acne vulgaris effects ( Figure 7). Since these four (4) genes were overexpressed in Acne vulgaris (Table 2), inhibitors will be needed as a treatment.

Molecular Docking Analysis
Last but not least, we used molecular docking to assess the likelihood of binding between the four selected targets and the screened phytocompounds from neem oil (Figure 8). According to earlier research, a binding affinity of −4.25 kcal/mol meant that the two molecules could bind with average efficiency, −5.0 kcal/mol meant good binding, and −7.0 kcal/mol meant strong binding activity [11]. Each of the 4 identified targets and the 10 screened phytocompounds from neem oil were docked in our analysis. The related hits (indicated by their PubChem IDs) for STAT1, CSK, CRABP2, and SYK have the binding affinities of ID_610088 (−7.9 kcal/mol), ID_16451547 (−7.6 kcal/mol), ID_16451547 (−8.5 kcal/mol), and ID_600826, respectively.

Molecular Docking Analysis
Last but not least, we used molecular docking to assess the likelihood of binding between the four selected targets and the screened phytocompounds from neem oil ( Figure  8). According to earlier research, a binding affinity of −4.25 kcal/mol meant that the two molecules could bind with average efficiency, −5.0 kcal/mol meant good binding, and −7.0 kcal/mol meant strong binding activity [11]. Each of the 4 identified targets and the 10 screened phytocompounds from neem oil were docked in our analysis. The related hits (indicated by their PubChem IDs) for STAT1, CSK, CRABP2, and SYK have the binding affinities of ID_610088 (−7.9 kcal/mol), ID_16451547 (−7.6 kcal/mol), ID_16451547 (−8.5 kcal/mol), and ID_600826, respectively. By examining the precise binding sites and the spatial distance in Discovery Studio, the outcomes of the docking between the hits from the ten (10) screened neem oil compounds and their respective targets were visualized. The findings revealed that STAT1 (PDB ID: 1yvl), ID_610088, has five (5) hydrophobic interactions involving the amino acid residues ALA246, CYS247, ARG321, PRO332, and LEU256, and two (2) hydrogen bonds with the amino acid residues of GLU320 and CYS324, with distances of 2.9886 and 4.1818 Å, respectively ( Figure 9; Table 3). In CSK (PDB ID: 1qpe), ID_16451547 had nine (9) hydrophobic interactions involving ALA381, VAL259, MET292, VAL301, PHE383, LEU251, ALA271, LEU371, and LYS273 amino acid residues. The distance between these contacts was 3.42564 Å. (Figure 10; Table 4). In CRABP2 (PDB ID: 1cbs), ID_16451547 has seven hydrophobic interactions involving the amino acid residues, ALA32, ILE63, VAL76, MET123, ALA36, and ARG59 and one hydrogen bond with the amino acid residue ARG111, the distance being 3.50388 Å. (Figure 11; Table 5). In SYK (PDB ID: 1xbc), ID_600826 has eight hydrophobic interactions involving LEU377, MET448, PRO455, LEU501, VAL385, LYS402, ALA400, and MET450 amino acid residues. The distance between these contacts was 2.84223 and 3.54405 Å, respectively ( Figure 12; Table 6). By examining the precise binding sites and the spatial distance in Discovery Studio, the outcomes of the docking between the hits from the ten (10) screened neem oil compounds and their respective targets were visualized. The findings revealed that STAT1 (PDB ID: 1yvl), ID_610088, has five (5) hydrophobic interactions involving the amino acid residues ALA246, CYS247, ARG321, PRO332, and LEU256, and two (2) hydrogen bonds with the amino acid residues of GLU320 and CYS324, with distances of 2.9886 and 4.1818 Å, respectively ( Figure 9; Table 3). In CSK (PDB ID: 1qpe), ID_16451547 had nine (9) hydrophobic interactions involving ALA381, VAL259, MET292, VAL301, PHE383, LEU251, ALA271, LEU371, and LYS273 amino acid residues. The distance between these contacts was 3.42564 Å. (Figure 10; Table 4). In CRABP2 (PDB ID: 1cbs), ID_16451547 has seven hydrophobic interactions involving the amino acid residues, ALA32, ILE63, VAL76, MET123, ALA36, and ARG59 and one hydrogen bond with the amino acid residue ARG111, the distance being 3.50388 Å. (Figure 11; Table 5). In SYK (PDB ID: 1xbc), ID_600826 has eight hydrophobic interactions involving LEU377, MET448, PRO455, LEU501, VAL385, LYS402, ALA400, and MET450 amino acid residues. The distance between these contacts was 2.84223 and 3.54405 Å, respectively ( Figure 12; Table 6).                Using the drawprotein package in R [12], protein chains, domains, regions, motifs, or phosphorylation sites were sketched to better understand the mechanism of binding between the protein targets and the phytocompounds from neem oil. LCK is a c-src tyrosine kinase (CSK) subunit [13]. The protein kinase domain of LCK contained GLY322, which was engaged in the hydrogen bond between the hit molecule, ID_16451547, and LCK in this protein target. Additionally, the same area was the site of all hydrophobic interactions  Using the drawprotein package in R [12], protein chains, domains, regions, motifs, or phosphorylation sites were sketched to better understand the mechanism of binding between the protein targets and the phytocompounds from neem oil. LCK is a c-src tyrosine kinase (CSK) subunit [13]. The protein kinase domain of LCK contained GLY322, which was engaged in the hydrogen bond between the hit molecule, ID_16451547, and LCK in this protein target. Additionally, the same area was the site of all hydrophobic interactions involving the molecules ALA381, VAL259, MET292, VAL301, PHE383, LEU251, ALA271, LEU371, and LYS273 ( Figure 13). The hit molecule, ID_600826, and SYK formed hydrogen bonds involving the residues LEU377 and ASP512 in the protein's interdomain B and protein kinase region. Additionally, the same area was the site of all hydrophobic interactions involving LEU377, MET448, PRO455, LEU501, VAL385, LYS402, ALA400, and MET450 ( Figure 14).

Discussion
Neem oil is an excellent herbal skincare ingredient as it has antimicrobial and antiinflammatory properties, as well as the ability to support the immune system [14,15]. It can fight with bacteria to prevent the recurrence of acne lesions and makes the skin healthy [16].
The results of this study reveal that the ten (10) neem oil ligands that were evaluated as possible medications for the gene products of STAT1, CSK, CRABP2, and SYK are promising therapeutic targets.
Without the signal transducer and activator of transcription (STAT) protein, the The hit molecule, ID_600826, and SYK formed hydrogen bonds involving the residues LEU377 and ASP512 in the protein's interdomain B and protein kinase region. Additionally, the same area was the site of all hydrophobic interactions involving LEU377, MET448, PRO455, LEU501, VAL385, LYS402, ALA400, and MET450 ( Figure 14). The hit molecule, ID_600826, and SYK formed hydrogen bonds involving the residues LEU377 and ASP512 in the protein's interdomain B and protein kinase region. Additionally, the same area was the site of all hydrophobic interactions involving LEU377, MET448, PRO455, LEU501, VAL385, LYS402, ALA400, and MET450 ( Figure 14).

Discussion
Neem oil is an excellent herbal skincare ingredient as it has antimicrobial and antiinflammatory properties, as well as the ability to support the immune system [14,15]. It can fight with bacteria to prevent the recurrence of acne lesions and makes the skin healthy [16].
The results of this study reveal that the ten (10) neem oil ligands that were evaluated as possible medications for the gene products of STAT1, CSK, CRABP2, and SYK are promising therapeutic targets.
Without the signal transducer and activator of transcription (STAT) protein, the

Discussion
Neem oil is an excellent herbal skincare ingredient as it has antimicrobial and antiinflammatory properties, as well as the ability to support the immune system [14,15]. It can fight with bacteria to prevent the recurrence of acne lesions and makes the skin healthy [16].
The results of this study reveal that the ten (10) neem oil ligands that were evaluated as possible medications for the gene products of STAT1, CSK, CRABP2, and SYK are promising therapeutic targets.
Without the signal transducer and activator of transcription (STAT) protein, the IFN/JAK signaling pathway cannot function [17]. The STAT proteins contain the Nterminal domain, the coiled-coil domain, the DNA-binding domain, the alpha-helical linker domain, the SH2 domain, and the transactivation domain [18]. The SH2 domain is necessary for the production of tyrosine phosphodimers and receptor binding. Seven genes make up the STAT family: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. These individuals all play a part in immunological defense, surveillance, and homeostasis [19]. Although STAT1 was the first member of the STAT family to be identified, its function in Acne vulgaris is still not fully known. When a ligand is stimulated, receptor-activated kinases such as JAK first phosphorylate and activate STAT1 in the classical signaling pathway [20]. When STAT1 is phosphorylated, it joins forces with other STAT family members, such STAT3, to create homodimers or heterodimers that go from the cytosol to the nucleus, where they function as transcription factors [21]. Due to its critical function in the immune response and defense against pathogen infections, STAT1 is typically thought of as a tumor suppressor [22]. However, a number of illnesses, including Acne vulgaris, have been linked to aberrant STAT1 activity [23]. Although there has been progress in our understanding of STAT activation, little is known about how STAT signals are downregulated, which is consistent with our recommended therapeutic approaches [24]. The biological process will be controlled by inhibiting the overexpressed STAT1 during Acne vulgaris infection (GO:0001817; p-Value = 9.25 × 10 −7 ). In response to cytokine stimulation, dormant cytoplasmic transcription factors known as STAT proteins are tyrosine phosphorylated to activate them [25]. The target molecule from neem oil, ID_610088, had more hydrophobic contacts than hydrogen bonds to enable this therapeutic process (two hydrogen bonds with amino acid residues of GLU320 and CYS324, the distances were 2.9886 and 4.1818, respectively, and five hydrophobic interactions involving ALA246, CYS247, ARG321, PRO332, and LEU256 amino acid residues). Short-range attractive interactions known as hydrophobic interactions play a significant role in the binding affinities between ligands and receptors [26]. Drugs on the market typically contain 16 hydrophobic atoms, with one to two donors and three to four acceptors [27]. This explains the significance of hydrophobic interactions in the development of drugs. They may improve the affinity of the target drug surfaces for binding. It has already been mentioned that adding hydrophobic interactions at the region of the hydrogen bonding can increase binding affinity and medication efficacy [28].
The 13-cis retinoic acid can be isomerized to all trans retinoic acid (ATRA) by sebocytes [29]. Isotretinoin elevates cellular retinoid acid-binding protein-2 (CRABP-2) expression in sebocytes [30], which delivers ARTA to retinoic acid receptors (RARs) that control gene expression [31]. A TATA-box found in the CRABP2 gene promoter is quickly activated by ATRA via a retinoic acid response element (RARE) [32]. Patients taking isotretinoin have suprabasal sebocytes that have higher levels of CRABP-2 expression compared to the epidermis, which encourages the preferential transport of ATRA to sebocyte RARs [33]. ATRA binding to nuclear RARs increases the expression of key transcription factors involved in apoptosis, such as the forkhead box transcription factors FoxO1 and FoxO3a and TRAIL [34]. Agamia et al.'s research [35] showed that oral isotretinoin treatment enhanced the nuclear levels of FoxO1 and FoxO3a in the sebaceous glands of Acne vulgaris patients. The expression of p53 is enhanced by ATRA exposure in epidermal keratinocytes, as has been demonstrated [36]. Shi et al. [37] found that isotretinoin exposure increased the expression of p53, FoxO1, and p21 in human primary keratinocytes. As Melnik has proposed [38], sebocyte apoptosis and isotretinoin-mediated teratogenicity (neural crest cell death) may both be caused by isotretinoin-induced overexpression of p53 [39]. In actuality, p53 expression and death are induced in melanoma cells by both isotretinoin and ATRA [40]. However, CRABP2 overexpression speeds up the development of tumors [41].
For 20 years, researchers have studied thousands of women. They discovered that people who have severe acne as teenagers may be more susceptible to developing melanoma, a type of skin cancer. The androgen hormone is linked to both melanoma and acne. The most dangerous type of skin cancer, melanoma, is rare [42]. The preferred pharmaceutical strategy is to develop inhibitors that specifically target CRABP2, as melanoma is a danger in Acne vulgaris infection. The maximum binding energy is, predictably, possessed by ID_16451547 from neem oil, which is likewise distinguished by having more hydrophobic interactions than hydrogen bonds.
A series of intricate biological reactions known as inflammation are used to defend the host against pathogen invasion [43]. Acne vulgaris is a self-limiting, inflammatory disorder of the pilosebaceous unit that is persistently recurrent. In teenagers with dehydroepiandrosterone naturally present in the bloodstream, Propionibacterium acnes develops Acne vulgaris [44]. It is well known that Syk, a cytoplasmic protein-tyrosine kinase, links immune cell receptors to intracellular signaling pathways that regulate cellular reactions to external antigens and antigen-immunoglobulin complexes, which are particularly important for the onset of inflammatory responses. Because Syk is a desirable target, therapeutic kinase inhibitors designed to lessen the symptoms and consequences of both acute and chronic inflammation are effective [45]. Thus, it was found that Syk is a key mediator in the immunological dysfunction brought on by inflammation [46]. In addition, we propose the neem oil compound ID_600826 as a viable therapeutic candidate for the management of Acne vulgaris by regulating Syk activity. Higher hydrophobic interactions than hydrogen bonds are another characteristic of this interaction.
The hits were connected to the targets' protein kinase domains, according to domain analyses for the SYK and CSK proteins. A crucial element required for the catalytic actions carried out by protein kinases is the protein kinase domain [47,48]. This further implies that the hit molecules' modulatory function takes place within the protein kinase domains of both kinases (SYK and CSK).

Collection and Handling of Plant Sample
The Department of Plant Biology Herbarium, University of Ilorin, gathered, identified, and validated the plant material, Azadirachta indica leaves. The voucher number was assigned as UILH/002/979/2023. The leaves were broken down into tiny bits and stored for extraction in a dry glass jar.

Extraction of Neem Oil by Steam Distillation
The oil from the neem leaves was extracted using the steam distillation process. The leaves were arranged in a column, the bottom of which was attached to a flask containing hot water. The upper portion was attached to a condenser, where the oils were transported to the condenser by the steam that was produced as it traveled through the leaves.

GC-MS Analysis of Oil
The phytochemical composition of the oil was ascertained using a gas chromatography from Agilent USA coupled to a mass spectrophotometer analyzer (5975C) JEOL (Freising, Germany) with triple axis detector and an auto injector (10-L syringe). As a carrier gas, helium gas was used at a constant pressure mode of 16.2 psi. On a capillary column (30 m × 250 m × 0.2 m) coated with phenyl methyl siloxane, all of the chromatographic separation was carried out. Ion source temperature (EI), interface temperature, and out time of 1.8 mm were additional GC-MS requirements. With a split ratio of 1:50 and an injection temperature of 280 • C, 1 L was injected in split mode. The column temperature increased from 50 to 100 • C at a rate of 20 • C/min, then to 250 • C at a rate of 20 • C/min, where it remained for 5 min. It took 19 min to elute completely. The system was managed by Ms Solution software, which was also used to examine the GC-MS data. By matching the obtained mass spectra with the standard NIST mass spectral database (NIST v20), the oil's identification was completed.

Drug-Likeness Analysis
For the evaluation of drug-likeness based on Lipinski's rule, the DataWarrior v5.5.0 program was utilized [49]. Molecules' 2D structures were downloaded in a structure data file format from PubChem and entered in DataWarrior for evaluation.

Target Prediction of Neem oil against Acne vulgaris
To forecast the probable targets of phytocompounds from neem oil, the reverse pharmacophore localization database PharmMapper (http://www.lilab-ecust.cn/pharmmapper/ (accessed on 4 January 2023)) [50] was employed. The SDF structure formats of these small molecular compounds were downloaded from the PubChem database (https:// pubchem.ncbi.nlm.nih.gov/ (accessed on 4 January 2023)) and uploaded to the PharmMapper server once the active components from the DataWarrior had been screened. Uniprot (https://www.uniprot.org/ (accessed on 4 January 2023)) [51] chose the species "Homo sapiens" to standardize the Uniprot ID to the gene symbol. Targets for Acne vulgaris were predicted in the Gene Cards database (https://www.genecards.org/ (accessed on 4 January 2023)) at the same time. To gather possible genes, the term "acne" was utilized. For additional analysis, we chose targets for Acne vulgaris with correlation scores > 1, targets for neem oil components with norm fits > 0.6, and the overlapped area of the Venn diagram, which showed possible targets for neem oil anti-Acne vulgaris.

Microarray Datasets
A functional genomics database open to the public called GEO (http://www.ncbi.nlm. nih.gov/geo (accessed on 15 January 2023)) contains a range of data, including information obtained through microarrays and next-generation sequencing. Keywords such as "Acne vulgaris" [MeSH terms] AND "Homo sapiens" [porgn] AND "gse" [filter] AND "profile expression per matrix" [filter] AND "feature name texture" [filter] were used to search the GEO database. After that, GSE6475, a single gene expression profile, was gathered. The GEO database provided the microarray data. The Affymetrix Human Genome U133A 2.0 microarray served as the foundation for GSE6475.

Data Pre-Processing and Identification of Differentially Expressed Genes (DEGs)
The GEO database's table array file for GSE6475 was downloaded. The dataset's probes were changed into common gene symbols before analysis. The dataset was normalized using NormExpression package in R software, version 4.0.0 (www.R-project.org (accessed on 20 January 2023)), and normalization was performed separately for each gene expression dataset. NormExpression provides a framework and a fast and simple way for researchers to select the best method for the normalization of their gene expression data based on the evaluation of different methods (particularly some data-driven methods or their own methods) in the principle of the consistency of metrics and the consistency of datasets [52]. The normalization was based on robust multi-array averaging. Microarray gene expression data can be seen and statistically analyzed using the GEO2R platform [53]. Using GEO2R software, DEGs from each dataset were identified in the current investigation. The threshold was established at a p-value of 0.05. The ggplo2 package in R software was then used to create a volcano plot of DEGs from the dataset.

Functional and Pathway Enrichment Analysis
Gene ontology (GO) analysis is a prominent technique used for extensive functional investigations of transcriptomic data and genomic data analysis. A database for the systematic examination of gene functions is the Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/pathway (accessed on 20 January 2023)) [54]. Large lists of genes or proteins can be systematically evaluated for their biological importance using the Integrated Annotation and Discovery Visualization Database (DAVID; https: //david.ncifcrf.gov (accessed on 4 January 2023)) [55]. The identified DEGs and genes in the significant parts of the current study were subjected to GO function and KEGG pathway enrichment analysis using DAVID. p values 0.005 were used to determine the significance of terms.

Pathway Analysis and Protein-Protein Interaction Analysis
A network-based method for displaying and deciphering gene array enrichment is an enrichment map [56]. The Enrichment Map program (http://www.baderlab.org/Software/ EnrichmentMap (accessed on 20 January 2023)) was used to conduct pathway intersection analysis in order to extract interactions between significantly enhanced signaling pathways. Silver was defined as a Benjamini-Hochberg adjusted p-value 0.05. Two indices for assessing sample group similarity are the Jaccard coefficient and the overlap coefficient.
A database of protein-protein interactions (PPIs) for 5090 species is available in STRING version 11.0. Protein-protein interactions, including direct (physical) and indirect (functional) linkages, can be accessed via the STRING database. PPIs that possessed at least a medium confidence score of 0.400 were considered for network generation.
Through the use of STRING and function and pathway enrichment analysis, a PPI network was created in order to evaluate the relationships between the DEGs discovered in this study. At P 0.05, this is deemed statistically significant.

Ligand Selection and Preparation:
The downloaded 2D structure in MOL SDF format was converted to a PDBQT file using PyRx tool to generate atomic coordinates and energy was minimized by optimization using the optimization algorithm at force field set at mmff94 (required) on PyRx ( Figure 15A-D). pathways. Silver was defined as a Benjamini-Hochberg adjusted p-value 0.05. Two indices for assessing sample group similarity are the Jaccard coefficient and the overlap coefficient.
A database of protein-protein interactions (PPIs) for 5090 species is available in STRING version 11.0. Protein-protein interactions, including direct (physical) and indirect (functional) linkages, can be accessed via the STRING database. PPIs that possessed at least a medium confidence score of 0.400 were considered for network generation.
Through the use of STRING and function and pathway enrichment analysis, a PPI network was created in order to evaluate the relationships between the DEGs discovered in this study. At P 0.05, this is deemed statistically significant.

Ligand Selection and Preparation:
The downloaded 2D structure in MOL SDF format was converted to a PDBQT file using PyRx tool to generate atomic coordinates and energy was minimized by optimization using the optimization algorithm at force field set at mmff94 (required) on PyRx (Figure 15A-D).

Accession and Preparation of the Target Protein
The target proteins, STAT1, CSK, CRABP2, and SYK were prepared by retrieving their corresponding three-dimension crystal structures from RCSB PDB (http://www.rcsb.org/pdb/home/home.do (accessed on 24 January 2023)) [57]. Subsequently, the bound complex molecules with the proteins were removed. The non-essential water molecules and all heteroatoms were removed using Pymol tool and Discovery stu-

Accession and Preparation of the Target Protein
The target proteins, STAT1, CSK, CRABP2, and SYK were prepared by retrieving their corresponding three-dimension crystal structures from RCSB PDB (http://www.rcsb.org/ pdb/home/home.do (accessed on 24 January 2023)) [57]. Subsequently, the bound complex molecules with the proteins were removed. The non-essential water molecules and all heteroatoms were removed using Pymol tool and Discovery studio 2017R2, respectively.

Molecular Docking
The molecular docking between the active ingredients in neem oil and the chosen target for the therapy of Acne vulgaris was examined using PyRx Autodock Vina. By thoroughly analyzing the resolution and release time in the Protein Data Bank (PDB) (www.rcsb.org (accessed on 24 January 2023)) [57] website, target proteins were discovered. The Discovery studio program was used to establish the precise binding locations and atomic distances between proteins and active substances.

Conclusions
Neem oil contains a number of active constituents that can be effective in the management of Acne vulgaris via different mechanisms. As seen from this research, STAT1, CSK, CRABP2, and SYK genes play pivotal roles in the pathophysiology of acne and thus serve as useful targets for neem oil. Via CRABP-2, neem oil is not only able to manage acne but also prevent the future possibility of melanoma. Based on the number of hydrophobic interactions, ID_610088 (2-(1-adamantyl)-N-methylacetamide), ID_600826 (N-benzyl-2-(2-methyl-5-phenyl-3H-1,3,4-thiadiazol-2-yl)acetamide), and ID_16451547 (N-(3-methoxyphenyl)-2-(1-phenyltetrazol-5-yl)sulfanylpropanamide) show efficacy against these target genes, thus concluding that neem oil possesses activity for the management of Acne vulgaris.